Capturing Protein Dynamics with Time-Resolved Luminescence Spectroscopy

The presented doctoral research utilizes time-resolved spectroscopy to characterize protein dynamics and folding mechanisms. We resolve millisecond-timescale folding by coupling time-resolved fluorescence energy transfer (trFRET) to a continuous flow microfluidic mixer to obtain intramolecular distance distributions throughout the folding process. We have elucidated the folding mechanisms of two cytochromes---one that exhibits two-state folding (cytochrome cb562) and one that has both a kinetic refolding intermediate ensemble and a distinct equilibrium unfolding intermediate (cytochrome c552). Our data reveal that the distinct structural features of cytochrome c552 contribute to its thermostability.;We have also investigated intrachain contact dynamics in unfolded cytochrome cb562 by monitoring electron transfer, which occurs as the heme collides with a ruthenium photosensitizer, covalently bound to residues along the polypeptide. Intrachain diffusion for chemically denatured proteins proceeds on the microsecond timescale with an upper limit of 0.1 microseconds. The power-law dependence (slope = -1.5) of the rate constants on the number of peptide bonds between the heme and Ru complex indicate that cytochrome cb562 is minimally frustrated.